Compounds and their salts specific to the PPAR receptors and the EGF receptors and their use in the medical field

ABSTRACT

The present invention relates to compounds comprising the general formula (I), in which R 1  and R 2 , which may be identical or different, are selected from the group comprising —H or a linear or branched alkyl group having from 1 to 6 carbon atoms or together form an aromatic or aliphatic ring with 5 or 6 atoms; Y and Z, which may be identical or different, are selected from the group comprising —H, —OH, —COOH, —OR 3 , —CH(OR 3 )COOH, in which R 3  is selected from H, phenyl, benzyl, —CF 3  or —CF 2 CF 3 , vinyl, allyl and a linear or branched alkyl group having from 1 to 6 carbon atoms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/671,579, filed Mar. 27, 2015, which is a divisional of U.S.application Ser. No. 14/202,386, filed Mar. 10, 2014 (issued as U.S.Pat. No. 9,133,099), which is a continuation of U.S. application Ser.No. 13/408,439, filed Feb. 29, 2012 (issued as U.S. Pat. No. 8,710,100),which is a divisional of U.S. application Ser. No. 11/989,090, filedJun. 6, 2008 (issued as U.S. Pat. No. 8,153,841), which is the nationalphase of International (PCT) Application No. PCT/IE2006/000078, filedJul. 24, 2006, which claims the benefit of and priority to ItalianPatent Application No. RM2005 A 000389, filed Jul. 22, 2005, the entiredisclosure of each of which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to compounds and their salts specific tothe PPAR receptors and the EGF receptors and their use in the medicalfield.

OBJECT OF THE INVENTION

In particular, the compounds and their salts according to the presentinvention can be used advantageously for the prevention and treatment oftumours expressing the PPARγ receptors (PeroxisomeProliferator-Activated Receptors) and the EGF receptors (EpidermalGrowth Factor receptors) such as tumours of the: oesophagus, stomach,pancreas, colon, prostate, breast, uterus and appendages, kidneys andlungs. Moreover, the compounds and their salts according to theinvention can be used for the treatment of chronic inflammatorydiseases, in particular chronic intestinal diseases, such as Crohn'sdisease and ulcerative rectocolitis.

BACKGROUND TO THE INVENTION

The PPARγ receptors are nuclear receptors (group of approx. 50transcription factors) which control the expression of many genes thatare important for the regulation of lipid metabolism, the synthesis ofinsulin and the processes of carcinogenesis and inflammation. (Bull A W,Arch Pathol Lab Med 2003; 127: 1121-1123) (Koeffler H P, Clin Cancer Res2003; 9: 1-9) (Youssef J et al., J Biomed Biotec 2004; 3: 156-166).

There are various natural and synthetic agonists which bind to the PPARγreceptors and alter their conformation, giving rise to activation.Natural and synthetic ligands are described in The Lancet 2002;360:1410-1418.

Recent studies have shown that treatment of tumour cells with ligands ofthe PPARγ receptors induces a decrease in cellular proliferation, celldifferentiation and apoptosis, suggesting potential application of suchcompounds as agents for preventing carcinogenesis (Osawa E et al.,Gastroenterology 2003; 124:361-367).

Other studies have shown that ligands of the PPARγ receptors (e.g.troglitazone) have anti-inflammatory effects and inhibit the mucosalinflammatory response in animal models of IBD (Tanaka T at al., CancerRes 2001; 61: 2424-2428).

Moreover, evidence has been published very recently that the intestinalanti-inflammatory activity of 5-ASA, the gold standard in the treatmentof IBD, is dependent on binding, and consequent activation, of the PPARγreceptors (Rousseaux C at al., J Exp Med 2005; 201: 1205-1215).

The transmembrane receptor with tyrosine-kinase EGF activity isexpressed to a very high degree in activated form in various types ofneoplasms (Mendelsohn J, Endocr Relat Cancer 2001; 8: 3-9) (Harari P M,Endocr Relat Cancer 2004; 11: 689-708).

Overexpression of the receptor is also related to potential ability ofcarcinomatous cells to metastasize. In connection to this, it has beendemonstrated that EGF promotes the migration and invasiveness of variouscell types connected with lesions at the level of interactions with theextracellular matrix (Brunton et al., Oncogene 1997; 14: 283-293).

Numerous studies performed both on experimental animals and in humanshave established the efficacy of inhibitors of the EGF receptor incontrolling proliferation and the spread of tumours (Mendelsohn J,Endocr Relat Cancer 2001; 8: 3-9) (Harari P M, Endocr Relat Cancer 2004;11: 689-708).

There is no doubt that the intracellular signals triggered by activationof the EGF receptor facilitate the growth and survival of neoplasticcells, contributing to the development of the pathology, and that suchsignals are essential in determining the ability of tumour cells tospread and colonize remote organs.—(Mendelsohn J. Endocr Relat Cancer2001; 8: 3-9) (Kari C et al., Cancer Res 2003; 63: 1-5).

From the foregoing and bearing in mind, moreover, that from thebiological standpoint, chronic inflammatory processes play a part incarcinogenesis, it becomes clear that there is a real need forinnovative research into new chemical entities which, by theircomplementary action both on the PPARγ receptors and on the EGFreceptors, are able to exert anti-inflammatory and anti-tumour action,of the chemo-preventive, anti-proliferative and anti-metastatic type.

The present invention provides a novel class of compounds that aresuitable for the prevention and treatment of cancer and of chronicinflammation by the modulation of specific receptors such as the PPARγreceptors and the EGF receptors.

SUMMARY OF THE INVENTION

The present invention relates to novel and inventive medical andtherapeutic uses of a series of compounds in so far as any of thesecompounds are not known, the invention also relates to these compounds.

The present invention relates to compounds comprising the generalformula (I)

in which

R₁ and R₂, which may be identical or different, are selected from thegroup comprising —H or a linear or branched alkyl group having from 1 to6 carbon atoms or together form an aromatic or aliphatic ring with 5 or6 atoms;

Y and Z, which may be identical or different, are selected from thegroup comprising —H, —OH, —COOH, —OR₃, —CH(OR₃)COOH, in which R₃ isselected from H, phenyl, benzyl, —CF, or —CF₂CF₃, vinyl, allyl and alinear or branched alkyl group having from 1 to 6 carbon atoms.

The present invention also relates to a subgroup of compounds, ofgeneral formula (Ia)

in which

R₁ and R₂, which may be identical or different, are selected from thegroup comprising —H or a linear or branched alkyl group having from 1 to6 carbon atoms

Y and Z, which may be identical or different, are selected from thegroup comprising —H, —OH, —COOH, —OR₂, —CH(OR₃)COOH, in which R₃ isselected from —H and a linear or branched alkyl group having from 1 to 6carbon atoms.

In some embodiments of the Invention, Z and Y are different. In someembodiments of the invention, at least one of Y or Z terminates in—COOH. Therefore, in some embodiments of the invention, Y or Z (and insome embodiments at least one of Y or Z, and in some embodiments, onlyone of Y or Z) is —COOH. In some embodiments of the invention, Y or Z(and in some embodiments at least one of Y or Z, and in someembodiments, only one of Y or Z) is —CH(OR₃)COOH.

The present invention also relates to compounds according to bothformula (I) and (Ia), except wherein Y and Z, which may be identical ordifferent, are selected from the group comprising —H, —COOH, —OR₃,—CH(OR₃)COOH. Therefore, in some embodiments of the invention, Z or Ymay not be —OH. In such embodiments of the invention, compounds 10 and11 are excluded.

In some embodiments of the invention, when Y is —H and Z is —CH(OH)COOH,the group NR₁R₂ is connected at the 3′ position. Thus, in someembodiments of the invention, compound 21 is excluded:

In other embodiments of the invention, when Z is —OCH₃ and Y is —COOH,the group NR₁R₂ is connected at the 4′ position. Thus, in someembodiments of the invention, the compound 22 is excluded.

In some embodiments of the invention, when Y is —H and Z is—CH(OCH₃)COOH, the group NR₁R₂ is connected at the 4′ position. Thus, insome embodiments of the invention, compound 35 is excluded:

In particular, the aforementioned linear or branched alkyl group havingfrom 1 to 6 carbon atoms can be selected from —CH₃, —CH₂CH₃, —CH(CH₃)₂.—CH₂CH₂CH₃, —C_(n)H_(2n-1).

The compounds of formula (I) and (Ia) can be selected from the groupcomprising

-   3-(3′-aminophenyl)2-hydroxypropanoic acid (compound 20)-   2-(4-aminophenyl)2-methoxyacetic acid (compound 23)-   2-(3-aminophenyl)2-ethoxyacetic acid (compound 32)-   2-(4-aminophenyl)2-ethoxyacetic acid (compound 33)-   3-(4′-aminophenyl)2-methoxypropionic acid (compound 34)-   3-(4′-aminophenyl)2-ethoxypropionic acid (compound 39)-   3-(3′-aminophenyl)2-ethoxypropionic acid (compound 40).

The above compound names can also be written in standard chemicalnomenclature as follows (which nomenclature will be used throughout thetext):

-   (±)-2-hydroxy-3-(3′-aminophenyl) propionic acid (compound 20)-   (±)-2-methoxy-2-(4′-aminophenyl) acetic acid (compound 23)-   (±)-2-ethoxy-2-(3′-aminophenyl) acetic acid (compound 32)-   (±)-2-ethoxy-2-(4′-aminophenyl) acetic acid (compound 33)-   (±)-2-methoxy-3-(4′-aminophenyl) propionic acid (compound 34)-   (±)-2-ethoxy-3-(4′-aminophenyl) propionic acid (compound 39)-   (±)-2-ethoxy-3-(3′-aminophenyl) propionic acid (compound 40).

The compounds according to the present invention cart be usedadvantageously in the medical field. Therefore the present inventionrelates to a pharmaceutical composition comprising one or more compoundsas defined above as active principles in combination with one or morepharmaceutically acceptable excipients or adjuvants.

The present invention relates, moreover, to the use of the compounds asdefined above for the preparation of a medicinal product for theprevention and treatment of tumours expressing the PPARγ receptors andthe EGF receptors such as, for example, tumour of the oesophagus,stomach, pancreas, colon, prostate, breast, of the uterus and itsappendages, of the kidneys and of the lungs.

Moreover, the invention relates to the use of the compounds according tothe invention for the preparation of a medicinal product for thetreatment of chronic inflammatory diseases such as, for example, Crohn'sdisease and ulcerative rectocolitis.

In particular, the compounds according to the invention that can be usedin the aforementioned applications, apart from those already described,can be as follows:

-   (R,S)-2-hydroxy-2-(3-aminophenyl)acetic acid (compound 10)-   (R,S)-2-hydroxy-2-(4-aminophenyl)acetic acid (compound 11)-   (R,S)-2-hydroxy-3-(4′-aminophenyl)propionic acid (compound 21)-   (R,S)-2-methoxy-3-(3′-aminophenyl)propionic acid (compound 35)-   (R,S)-2-methoxy-3-(3-aminophenyl)propionic acid (compound 34).

The above compound names can also be written in standard chemicalnomenclature as follows (which nomenclature will be used throughout thetext):

-   (±)-2-hydroxy-2-(3′-aminophenyl)acetic acid (compound 10)-   (±)-2-hydroxy-2-(4′-aminophenyl)acetic acid (compound 11)-   (±)-2-hydroxy-3-(4′-aminophenyl)propionic acid (compound 21)-   (±)-2-methoxy-3-(3′-aminophenyl)propionic acid (compound 35)-   (±)-2-methoxy-3-(4′-aminophenyl)propionic acid (compound 34).

According to one embodiment, R₃ of the compounds of formula (I) can be Haccording to the following formula (II)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₃ according to the following formula (III)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₂CH₃ according to the following formula (IV)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₂CH₃ according to the following formula (V)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₃ according to the following formula (VI)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₃ according to the following formula (VI)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₂CH₃ according to the following formula (VII)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₂CH₃ according to the following formula (VIII)

while R₁, R₂, X and Y are defined above.

According to another embodiment, R₃ of the compounds of formula (I) canbe —CH₃ according to the following formula (IX)

while R₁, R₂, X and Y are defined above.

Preferably, the compounds of formula (I) can be selected from the groupcomprising

-   (±)-2-hydroxy-3-(3′-aminophenyl) propionic acid (compound 20)-   (±)-2-methoxy-2-(4′-aminophenyl) acetic acid (compound 23)-   (±)-2-ethoxy-2-(3′-aminophenyl) acetic acid (compound 32)-   (±)-2-ethoxy-2-(4′-aminophenyl) acetic acid (compound 33)-   (±)-2-methoxy-3-(4′-aminophenyl) propionic acid (compound 34)-   (±)-2-ethoxy-3-(4′-aminophenyl) propionic acid (compound 39)-   (±)-2-ethoxy-3-(3′-aminophenyl) propionic acid (compound 40).

The compounds according to the present invention can be usedadvantageously in the medical field. Therefore the present inventionrelates to a pharmaceutical composition comprising one or more compoundsas defined above as active principles in combination with one or morepharmaceutically acceptable excipients or adjuvants.

The present invention relates, moreover, to the use of the compounds asdefined above for the preparation of a medicinal product for theprevention and treatment of tumours expressing the PPARγ receptors andthe EGF receptors such as, for example, tumour of the oesophagus,stomach, pancreas, colon, prostate, breast, of the uterus and itsappendages, of the kidneys and of the lungs.

Moreover, the invention relates to the use of the compounds according tothe invention for the preparation of a medicinal product for thetreatment of chronic inflammatory diseases such as, for example, Crohn'sdisease and ulcerative rectocolitis. The present invention also relatesto methods of treatment of humans and/or mammals (including rodents,farm animals, domestic pets, mice, rats, hamsters, rabbits, dogs, cats,pigs, sheep, cows, horses).

In particular, the compounds according to the Invention that can be usedin the aforementioned applications, apart from those already described,can be as follows:

-   (±)-2-hydroxy-2-(3-aminophenyl)acetic acid (compound 10)-   (±)-2-hydroxy-2-(4-aminophenyl)acetic acid (compound 11)-   (±)-2-hydroxy-3-(4′-aminophenyl)propionic acid (compound 21)-   (±)-2-methoxy-3-(3′-aminophenyl)propionic acid (compound 35)-   (±)-2-methoxy-3-(4′-aminophenyl)propionic acid. (compound 34)

The uses of the compounds described is not restricted to their use inthe racemic form. This invention extends to the use of any describedcompounds in the enantiomerically pure R or S forms, or any mixture inwhich one enantiomer is in excess of the other, in any proportion.

In fact, docking studies performed Indicate that the S enantiomer to bemore active than the R enantiomer, although the pure R enantiomer doesshow activity.

The molecules of the present invention were derived from molecularmodeling work using mesalazine as a basis and all chemically feasiblevariations were evaluated in order to achieve the best score (affinityand activation of the receptor) in computer docking experiments.Consequently, it is believed that the compounds of the present inventionthat show comparable function and/or activity to mesalazine do sothrough similar biological pathways. It is believed that similarcharacteristics to mesalazine inherent in the molecules of the inventionconfer upon the molecules a similar activity in relation to the EGFpathway.

The examples provided herein are useful models for use in the predictionof the use of the compounds in the various medical fields alreadydiscussed. The models therefore provide valuable and meaningful resultsregardless of their mechanism of action.

In addition to the above mentioned compounds, the present inventionprovides for the use of the following compounds:

The present invention will now be described for purposes ofillustration, but without limiting it, according to its preferredembodiments, with particular reference to the diagrams in the appendeddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE

Table 1. Percentages of DLD-1 cell inhibition by graded doses (0.5-10mM) of the specified compounds. Cells were cultured in the presence orabsence of the compounds, and cell growth was then assessed by thecolormetric (BrdU) assay after 48 hours culture.

FIG. 1 shows the structures of compounds 20, 23, 32, 33, 34, 35, 39 and40.

FIG. 2: PPARγ activity by treatment with compounds.

FIG. 3 and FIG. 4: Effect of the specified substances on theproliferation of human colon carcinoma cell lines (i.e. HT29, HT115 andDLD1). Cells were treated with increasing concentrations of substances(0.5-10 mM)) for 48 hours and the proliferation was determined by usinga colorimetric assay for the measurement of BrdU Incorporation. Opticaldensity (OD) was determined at 450 nm using an ELISA reader. Dataindicate the mean±SD of 3 separate experiments.

FIG. 5: Docking of (R) Compound 34 to PPARγ receptor (amino acidresidues labeling and hydrogen bonding is shown).

FIG. 6: Docking of (S) Compound 34 to PPARγ receptor (amino acidresidues labeling and hydrogen bonding is shown).

FIG. 7: Docking of (R) Compound 35 to PPARγ receptor (amino acidresidues labeling and hydrogen bonding is shown).

FIG. 8: Docking of (S) Compound 35 to PPARγ receptor (amino acidresidues labeling and hydrogen bonding is shown).

FIG. 9: Docking of (R) Compound 39 to PPARγ receptor (amino acidresidues labeling and hydrogen bonding is shown)

FIG. 10: Docking of (S) Compound 39 to PPARγ receptor (amino acidresidues labeling and hydrogen bonding is shown).

FIG. 11: Docking of mesalamine to PPARγ receptor (amino acid residueslabeling and hydrogen bonding is shown).

FIG. 12A and FIG. 12B: Schematic Synthesis and Subsequent Resolution ofCompound 39.

EXAMPLE 1 Method of Preparing (±)-2-hydroxy-3-(3′-aminophenyl)-propanoicAcid (Compound 20)

Step 1

3-Nitrobenzaldehyde (45.3 g, 0.3 mol), N-acetylglycine (42.1 g, 0.36mol) and sodium acetate (32 g, 0.39 mol) were mixed with aceticanhydride (142 ml, 1.5 mol) and the resulting mixture heated withstirring to 120° C. for 6 brs, giving a dark solution. The mixture wasthen cooled to RT overnight, resulting in the formation of aprecipitated solid. The reaction mixture was poured into ice-water (130g) and the resulting suspended solid was collected by filtration. Thecrude solid product (72 g) was washed with acetone (80 ml) thenrecrystallized from hot acetone (320 ml) to give a crystalline solidthat was washed with 50% aqueous ethanol, then dried at 40° C./40 mmHgto give 2-methyl-4-(3-nitrobenzylidene)oxazol-5(4H)-one (49.0 g, 78%) aspale yellow needles. ¹H NMR (δ, 250 MHz, CDCl₃)=2.47 (3H, s), 7.15 (1H,s), 7.63 (1H, dd, 8.2 & 7.6 Hz), 8.27 (1H, d, 8.2 Hz), 8.34 (1H, d, 7.6Hz), 9.02 (1H, s).

Step 2

2-Methyl-4-(3-nitrobenzylidene)oxazol-5(4H)-one (52.0 g, 0.224 mol) wasmixed with 3M hydrochloric acid (1.3 L) and the suspension stirred at100° C. for 6 h. The resulting suspension was stirred at RT overnightthen the suspended solid was collected by filtration, washed with water(2×40 ml), then dried in vacuo to give2-hydroxy-3-(3-nitrophenyl)acrylic acid (29.39). The combined filtrateand washes were extracted with ethyl acetate (4×0.5 L), then thecombined organic extracts were dried over sodium sulfate andconcentrated to dryness to give a further crop of2-hydroxy-3-(3-nitrophenyl)acrylic acid (12.0 g). The total yield of2-hydroxy-3-(3-nitrophenyl)acrylic acid was 41.3 g (88%),

¹H NMR (δ, 250 MHz, DMSO-d₆)=6.56 (1H, s), 7.64 (1H, t, 8 Hz), 8.0-8.1(2H, m), 8.78 (1H, s), 9.95 (1H, brs), 12.80 (1H, brs).

Step 3

Sodium ethoxide (1.8 g, 26.4 mmol) was added portionwise at 0° C. to astirred solution of 2-hydroxy-3-(3-nitrophenyl)acrylic acid (5.25 g,25.0 mmol) in methanol (131 ml) to form a clear, pale yellow solution.Sodium borohydride (1 g, 26.4 mmol) was then carefully added in twoportions and the mixture stirred at 5-10° C. for 30 mins. A small amountof water was then added to quench the reaction and destroy any excessNaBH₄. The methanol was removed in vacuo to give a solid residue, whichwas ground with a 5:2 mixture of ethyl acetate and heptane (21 ml) thenfurther ground with 3% aqueous methanol. The resulting solid wascollected by filtration and dried in vacuo to give2-hydroxy-3-(3-nitrophenyl)propionic acid (3.0 g, 57%).

¹H NMR (δ, 250 MHz, DMSO-d₆)=2.97 (1H, dd, 14 & 8.2 Hz), 3.15 (1H, dd,14 & 4.2 Hz), 4.23 (1H, dd, 8.2 & 4.2 Hz), 7.58 (1H, t, 8 Hz), 7.70 (1H,d, 8 Hz), 8.0-8.15 (2H, m).

Step 4

A mixture of 2-hydroxy-3-(3-nitrophenyl)propionic acid (3.0 g, 14.2mmol), methanol (129 ml) and 5% palladium on activated charcoal (600 mg,2 mol %) was hydrogenated at 10 psi H₂ atmosphere for 1 hr. The mixturewas then filtered through celite, the filter cake was washed withmethanol and the filtrates concentrated at 40° C. under high vacuum togive the product as a foamy solid. This was dissolved in water and thesolution freeze-dried to give (±)-2-hydroxy-3-(3′-aminophenyl)-propanoicacid (2.6 g, 100%) as a white solid.

¹H NMR (δ, 250 MHz, DMSO-d₆)=2.61 (1H, dd, 13.6 & 8.3 Hz), 2.81 (1H, dd,13.6 & 4.6 Hz), 4.09 (1H, dd, 8.25 & 4.6 Hz), 6.35-6.43 (2H, m), 6.45(1H, d, 1 Hz), 6.90 (1H, t, 7.6 Hz).

EXAMPLE 2 Method of Preparing (±)-2-methoxy-2-(4′-aminophenyl)-aceticAcid (Compound 23)

Step 1

A solution of potassium hydroxide (6.72 g, 0.12 mol) in methanol (25 ml)was added to a cooled (−7° C.) solution of 4-acetamidobenzaldehyde (24.5g, 0.15 mol) and chloroform (40.1 g, 0.33 moL) in DMF (100 ml) at such arate as to keep the temperature below 5° C. The mixture was allowed towarm to 2° C. over 5.5 h then it was added to a mixture of 1M aq. HCl(200 ml) and toluene (200 ml) and stirred overnight. The resulting2-(4-acetamidophenyl)-trichlorocarbinol was collected by filtration (29g) and suction dried.

Step 2

Solutions of 2-(4-acetamidophenyl)-trichlorocarbinol (14.0 g, 49.5 mmol)in methanol (330 ml) and potassium hydroxide (13.8 g, 250 mmol) inmethanol (150 ml) were combined and the mixture heated to 70-80° C. for3 hr. After cooling, the KCl by-product was removed by filtration thenconcentration of the filtrate in vacuo gave2-(4-acetamidophenyl)-2-methoxyacetic acid (14 g) as a white solid.

Step 3

2-(4-Acetamidophenyl)-2-methoxyacetic acid (7.1 g, 31.8 mmol) was heatedwith hydrazine monohydrate (40 ml) for 16 hr, cooled and concentrated invacuo. The resulting residual oil was purified by silica gel columnchromatography (eluent 20-40% methanol in CH₂Cl₂) to give 2.6 g (45%) of(±)-2-methoxy-2-(4′-aminophenyl)-acetic acid ¹H NMR (δ, 250 MHz, CD₃OD):3.27 (3H, s), 4.43 (1H, s), 6.66 (2H, d, 8.5 Hz), 7.18 (2H, d, 8.2 Hz).

EXAMPLE 3 Method of Preparing (±)-2-ethoxy-2-(3′-aminophenyl)-aceticAcid (Compound 32)

Step 1

3-Nitrobenzaldehyde (25 g, 165 mmol) and chloroform (30 ml, 375 mmol)were dissolved in DMF (100 ml) and the solution cooled to between −5° C.and −10° C. A fresh solution of potassium hydroxide (7.5 g, 134 mmol) inmethanol (22.5 ml) was added slowly so as maintain the internaltemperature <−5° C. The reaction was maintained at <−5° C. for 2 hr andthen quenched with a cooled mixture of aqueous hydrochloric acid (225ml) in toluene (225 ml). The solution was allowed to warm slowly to roomtemperature overnight in the ice bath. After this time the toluene layerwas separated and the aqueous layer further extracted with toluene. Thecombined organic layers were washed with water (2×225 ml), 5% sodiumbicarbonate solution (225 ml) and water (225 ml). The solution was dried(MgSO₄), filtered, and concentrated in vacuo to give2-(3-nitrophenyl)-trichlorocarbinol as an orange solid (42 g, 155 mmol,94%).

¹H NMR (δ, 250 MHz, CDCl₃): 3.7 (br, s, 1H), 5.4 (s, 1H), 7.6 (t, 1H,8.0 Hz), 8.0 (d, 1H, 8.0 Hz), 8.3 (d, 1H, 8.0 Hz), 8.5 (s, 1H).

Step 2

2-(3-Nitrophenyl)-trichlorocarbinol (20 g, 74 mmol) was dissolved inabsolute ethanol (74 ml) and a solution of potassium hydroxide (20.79,369 mmol) in absolute ethanol (150 ml) was added slowly. The solutionwas heated at reflux for 4 hr, allowed to cool and then concentrated invacuo. The residue was acidified with dilute hydrochloric acid and theproduct extracted in ethyl acetate (×3). The combined organic layerswere dried (MgSO₄), filtered, and concentrated in vacuo to give2-ethoxy-2-(3-nitrophenyl)acetic acid as a brown solid (6.4 g, 28.4mmol, 38%).

¹H NMR (5, 250 MHz, CD₃OD): 1.0 (t, 3H, 7.0 Hz), 3.8 (m, 1H), 3.7 (m,1H), 5.1 (s, 1H), 7.7 (t, 1H, 7.8 Hz), 7.9 (d, 1H, 7.8 Hz), 8.3 (d, 1H,7.8 Hz), 8.4 (s, 1H).

Step 3

2-Ethoxy-2-(3-nitrophenyl)acetic acid (6.4 g, 28.4 mmol) was dissolvedin absolute ethanol (500 ml), 5% palladium on carbon (wet) (1.5 g) addedand the mixture hydrogenated at 60 psi overnight. The suspension wasfiltered through celite and the filtrate concentrated to give(±)-2-ethoxy-2-(3′-aminophenyl)-acetic acid (3.0 g, 15.3 mmol, 54%) as abrown solid.

¹H NMR (δ, 250 MHz, CD₃OD): 1.2 (t, 3H, J=6.9 Hz), 3.5 (m, 1H), 3.6 (m,1H), 4.6 (s, 1H), 6.7 (d, 1H, J=7.6 Hz), 6.9 (m, 2H), 7.0 (t, 1H, J=7.6Hz).

EXAMPLE 4 Method of Preparing (±)-2-ethoxy-2-(4′-aminophenyl)-aceticAcid (Compound 33)

Step 1

See “Compound 23 step 1”

Step 2

Solutions of 2-(4-acetamidophenyl)-trichlorocarbinol (14.0 g, 49.5 mmol)in ethanol (400 ml) and potassium hydroxide (13.8 g, 250 mmol) inethanol (150 ml) were combined and the mixture heated at 70-80° C. for2.5 hr. The mixture was cooled, filtered to remove the KCl by-product,and concentrated in vacuo to give 2-(4-acetamidophenyl)-2-ethoxyaceticacid (14 g) as a yellow solid.

Step 3

2-(4-Acetamidophenyl)-2-ethoxyacetic acid (7.54 g, 31.8 mmol) was heatedwith hydrazine monohydrate (40 ml) for 16 hr, the mixture cooled thenconcentrated in vacuo. The residual oil was then purified by silicacolumn chromatography (20-40% methanol in CH₂Cl₂ eluent) to give(±)-2-ethoxy-2-(4′-aminophenyl)-acetic acid (2.3 g, 37%) as a whitefoam.

¹H NMR (δ, 250 MHz, CD₃OD): 1.18 (3H, t, 7.0 Hz), 4.42 (1H, qd, 7.3, 2.4Hz), 4.56 (2H, s), 5.50 (1H, qd, 7.0, 2.1 Hz), 6.66 (2H, d, 8.7 Hz),7.20 (2H, d, 8.5 Hz)

EXAMPLE 5 Method of Preparing (±)-2-methoxy-3-(4′-aminophenyl)-propionicAcid (Compound 34)

Step 1

4-Nitrobenzaldehyde (53.7 g, 0.356 mol), N-acetylglycine (49.9 g, 0.427mol) and sodium acetate (37.9 g, 0.463 mol) were mixed with aceticanhydride (168 g, 1.78 mol) and the resulting mixture heated withstirring to 120° C. for 6 hrs, giving a dark suspension. The mixture wasthen cooled to RT overnight, resulting in the formation of aprecipitated solid. The reaction mixture was poured into ice-water (150g) and the resulting suspended solid was collected by filtration. Thecrude solid product was washed with acetone (100 ml) then recrystallizedfrom hot acetone (650 ml) to give a crystalline solid that was washedwith 50% aqueous ethanol, then dried in vacuo to give2-methyl-4(4-nitrobenzylidene)oxazol-5(4H)-one (55.0 g, 66%) as paleyellow needles. The crystallisation mother liquors and washes werecombined and evaporated to give a solid residue that was recrystallizedfrom acetone to give a second crop of2-methyl-4-(4-nitrobenzylidene)oxazol-5(4H)-one (8 g, 10%). The combinedyield of 2-methyl-4-(4-nitrobenzylidene)oxazol-5(4H)-one was 63 g (76%)

¹H NMR (δ, 250 MHz, CDCl₃)=2.47 (3H, s), 7.14 (1H, s), 8.28 (4H, m).

Step 2

2-Methyl-4-(4-nitrobenzylidene)oxazol-5(4H)-one (63.0 g, 0.272 mol) wasmixed with 3M hydrochloric acid (1.2 L) and the suspension was stirredat 100° C. for 6 h. The resulting suspension was stirred at RT overnightthen the suspended solid was collected by filtration, washed with water(2×50 ml), then dried in vacuo to give2-hydroxy-3-(4-nitrophenyl)acrylic acid (46.6 g, 81%). The combinedfiltrate and washes were extracted with ethyl acetate (4×0.5 L), thenthe combined organic extracts were dried over sodium sulfate andconcentrated to dryness to get a further crop of2-hydroxy-3-(4-nitrophenyl)acrylic acid (0.8 g, 1%). The total yield of2-hydroxy-3-(4-nitrophenyl) acrylic acid was 47.4 g (82%).

¹H NMR (δ, 250 MHz, DMSO-d₆)=6.52 (1H, s) 8.01 (2H, d, 8.5 Hz), 8.22(2H, d, 8.5 Hz).

Step 3

A mixture of 2-hydroxy-3-(4-nitrophenyl)acrylic acid (15 g, 71.7 mmol),cesium carbonate (56 g, 172.1 mmol) and dimethyl sulphate (14.2 ml,150.6 mmol) in DMF (270 ml) was stirred at RT for 18 hr. Water (220 ml)and ethyl acetate (150 ml) were added and the layers separated. Theaqueous layer was further extracted with ethyl acetate (4×100 ml) thenthe combined organics were washed with water (6×100 ml), brine (2×120ml) and concentrated to half volume. Heptane was added (70 ml) and themixture concentrated to 200 ml volume. The resulting precipitated solidwas collected by filtration, washed with heptane (2×100 ml) and suctiondried on the filter to afford methyl 2-methoxy-3-(4-nitrophenyl)acrylateas a tan solid (9.2 g, 54% yield) containing a trace of heptane.

¹H NMR (δ, 250 MHz, DMSO-d₆): 3.82 (s, 3-H, OMe), 3.84 (s, 3-H, OMe),7.02 (s, 1-H. CH═), 8.04 (d, 2-H, CHaromatic), 8.26 (d, 2-H,CHaromatic).

Step 4

Methyl 2-methoxy-3-(4-nitrophenyl)acrylate (7.8 g, 32.8 mmol) wasdissolved in IMS (156 ml). A solution of NaOH (1.44 g, 36.1 mmol) inwater (78 ml) was added and the mixture stirred at ambient temperature(18° C.) for 18 hr. The reaction mixture was acidified with 1M HCl (120ml) and the resulting precipitated solid was collected by filtration,washed with water (2×100 ml) and partially suction dried on the filterfor 30 mins, followed by vacuum oven drying at 18° C. for 18 hr. Thus2-methoxy-3-(4-nitrophenyl)acrylic acid was afforded as a tan solidcontaining some water of crystallisation (6.7 g, 91%).

¹H NMR (δ, 250 MHz. DMSO-d₆): 3.83 (s, 3-H, OMe), 6.97 (s, 1-H, CH═),8.02 (d, 2-H, CHaromatic), 8.25 (d, 2-H, CHaromatic).

Step 5

2-Methoxy-3-(4-nitrophenyl)acrylic acid (6.7 g, 30 mmol) was taken up inmethanol (700 ml) and THF (300 ml) and 10% Pd on C (wet basis) (0.67 g)was added. The mixture was hydrogenated at 45 psi for 43 mins, followedby repeated refills to 45-48 psi every hour for 3 hr and finally 48 psifor 18 hr. The resulting suspension was filtered through GF/F filterpaper and the filter residue washed with MeOH (200 ml). The filtrateswere concentrated to an off-white solid. The solid was slurried in IMS(75 ml) at 20° C. for 1.5 hr, filtered, and washed with IMS/heptane(1:2) (20 ml) and dried on the filter for 1 hr to afford(±)-2-methoxy-3-(4′-aminophenyl)-propionic acid as an off-white solid(5.1 g, 88% yield)

¹H NMR (δ, 250 MHz, DMSO-d₆): 2.74 (m, 2-H), 3.23 (s, 3-H, CH), 3.80(dd, 1-H, CH), 6.47 (d, 2-H, aromatic), 6.87 (d, 2-H, aromatic).

EXAMPLE 6 Method of Preparing (±)-2-methoxy-3-(3′-aminophenyl)-propionicAcid (Compound 35)

Steps 1 & 2

As per compound 20

Step 3

Dimethyl sulfate (13.23 g, 105 mmol) was added to a stirred mixture of2-hydroxy-3-(3-nitrophenyl)acrylic acid (10.5 g, 50.0 mmol) and caesiumcarbonate (39.1 g, 120 mmol) in DMF (105 ml) to form a clear, paleyellow mixture, which was stirred at RT overnight. The resulting darkred suspension was concentrated in vacuo and the residue partitionedbetween water (100 ml) and dichloromethane (150 ml). The organic layerwas separated, further washed with water (2×100 ml), dried over sodiumsulphate and filtered through silica gel. The resulting yellow solutionwas evaporated to dryness in vacuo to give methyl2-methoxy-3-(3-nitrophenyl)acrylate as a yellow solid (8.1 g, 67%). ¹HNMR (δ, 250 MHz, DMSO-d₆)=3.81 (3H, s), 3.83 (3H, s), 7.08 (1H, s), 7.71(1H, dd, 7.9 & 8.2 Hz), 8.10-8.22 (2H, m), 8.66 (1H, s).

Step 4

A solution of potassium hydroxide (2.0 g, 35.9 mol) in water (25 ml) wasadded to a stirred solution of methyl2-methoxy-3-(3-nitrophenyl)acrylate (8.1 g, 34.2 mmol) in methanol (150ml) and the resulting mixture was stirred at RT overnight. A furtherquantity of KOH (0.5 g, 8.9 mmol) in water (10 ml) was added and themixture heated to 80° C. for 1 hr. The methanol was then evaporated invacuo and the residue diluted with water (200 ml). The solution waswashed with dichloromethane (2×100 ml), filtered through a pad of celiteand then acidified by the addition of 3M HCl to pH 3. The mixture wasrefrigerated for 18 h then the precipitated solid was collected byfiltration, washed with water (3×30 ml) and dried in vacuo at 40° C. togive 2-methoxy-3-(3-nitrophenyl)acrylic acid as a yellow solid (6.4 g,84%).

¹H NMR (δ, 250 MHz, DMSO-d₆)=3.82 (3H, s), 7.02 (1H, s), 7.70 (1H, t,7.93 Hz), 8.10-8.22 (2H, m), 8.65 (1H, s).

Step 5

A mixture of 2-methoxy-3-(3-nitrophenyl)acrylic acid (3.49, 15.25 mmol),methanol (340 ml) and 5% palladium on activated charcoal (1.36 g, 4 mol%) was hydrogenated at 12-36 psi H₂ atmosphere for 1.5 hr. The mixturewas then filtered through celite, the filter cake washed with methanoland the filtrates concentrated at 40° C. under vacuum to give theproduct as a foamy solid. This was dissolved in water (100 ml) and thesolution freeze-dried to give (±)-2-methoxy-3-(3′-aminophenyl)-propionicacid (2.6 g, 100%) as an off-white solid.

¹H NMR (δ, 250 MHz, DMSO-d₆)=2.68 (1H, dd, 13.9 & 8 Hz), 2.80 (1H, dd,13.9 & 4.6 Hz), 3.21 (3H, s), 3.84 (1H, dd, 8.25 & 4.6 Hz), 6.36-6.44(3H, m), 6.91 (1H, dd, 7.6 Hz).

EXAMPLE 7 Method of Preparing (±)-2-ethoxy 3-(4′-aminophenyl)-propionicAcid (Compound 39). Enantomeric Resolution (FIG. 12A and FIG. 12B)

Steps 1 & 2

As per compound 34 steps 1 & 2.

Step 3

2-Hydroxy-3-(4-nitrophenyl)acrylic acid (20 g, 95.6 mmol) was suspendedin DMF (200 ml). Cs₂CO₃ (74.9 g, 229.9 mmol) and diethyl sulphate (26.3ml, 201 mmol) were added and dissolution was observed. After stirringfor 18 hr at 18° C. water (350 ml) and ethyl acetate (250 ml) were addedand the layers separated. The aqueous layer was further extracted withethyl acetate (5×200 ml) then the combined organics were washed withwater (2×200 ml), brine (2×200 ml) and dried over magnesium sulfate. Theorganics were concentrated to dryness to obtain ethyl2-ethoxy-3-(4-nitrophenyl)-acrylate as an orange solid containing 3.6%by mass DMF (27.6 g wet, >100% yield). ¹H NMR (δ, 250 MHz, DMSO-d₆):1.32 (t, 6-H, 2×CH₂CH₃), 4.13 (q, 2-H, CH₂CH₃), 4.30 (q, 2-H, CH₂CH₃),6.99 (s, 1-H, CH═), 8.06 (d, 2-H, CHaromatic), 8.26 (d, 2-H,CHaromatic).

Step 4

Ethyl 2-ethoxy-3-(4-nitrophenyl)acrylate containing 3.6 wt % DMF (26.07g corrected, 98.3 mmol) was dissolved in IMS (500 ml) and a solution ofNaOH (1.44 g, 36.1 mmol) in water (260 ml) was added. The resultingmixture was stirred at ambient temperature for 18 hr then acidified with1M HCl (120 ml) and the resulting solid collected by filtration, washedwith water (2×100 ml) and suction dried on the filter for 30 mins,followed by vacuum oven drying at 18° C. for 18 hr,2-Ethoxy-3-(4-nitrophenyl)acrylic acid was thus obtained as an orangesolid containing water of crystallisation (18.4 g, 79%).

¹H NMR (δ, 250 MHz, DMSO-d₆): 1.31 (t, 3-H, Me), 4.11 (q, 2-H, CH₂),6.98 (e, 1-H, CH═), 8.05 (d, 2-H, CHaromatic), 8.25 (d, 2-H,CHaromatic).

Step 5

2-Ethoxy-3-(4-nitrophenyl)acrylic acid (18.4 g wet, approx. 77.5 mmol)was dissolved in MeOH (1.1 L) and 10% Pd on C (wet basis) (1.84 g) wasadded. The mixture was hydrogenated at 12 psi for 10 mins, followed byrepeated refill to 20-28 psi every 10-20 mins for 5 hr then 46 psi for18 hr. The mixture was filtered through GF/F paper and the residue wasslurried in IMS (100 ml), filtered, washed with heptane (100 ml), andsuction dried on the filter. Thus (±)-2-ethoxy3-(4′-Aminophenyl)-propionic acid was obtained as an off-white solid(11.2 g, 69%).

¹H NMR (δ, 250 MHz, DMSO-d₆): 1.03 (t, 3-H, CH₃), 2.73 (m, 2-H,), 3.29(m, 1H), 3.46 (m, 1H), 3.80 (dd, 1-H), 6.50 (d, 2-H), 6.87 (d, 2-H).

EXAMPLE 8 Method of Preparing (±)-2-ethoxy-3-(3′-aminophenyl)-propanoicAcid (Compound 40)

Steps 1 & 2

As per compound 20 steps 1 & 2.

Step 3

Diethyl sulfate (12 g, 78.2 mmol) was added to a stirred mixture of2-hydroxy-3-(3-nitrophenyl)acrylic acid (6.1 g, 30.0 mmol) and caesiumcarbonate (29.3 g, 90 mmol) in DMF (61 m) to form a clear, pale yellowmixture, which was stirred at RT overnight. The resulting dark redsuspension was heated to 50° C. for 4 h then concentrated in vacuo andthe residue partitioned between water (100 ml) and dichloromethane (150ml). The organic layer was separated, further washed with water (2×100ml), dried over sodium sulphate and filtered through a silica gel pad.The resulting yellow solution was evaporated to dryness in vacuo to giveethyl 2-ethoxy-3-(3-nitrophenyl)acrylate as a yellow solid (5.6 g, 72%).

Step 4

A solution of potassium hydroxide (1.3 g, 22.2 mol) in water (20 ml) wasadded to a stirred solution of ethyl 2-ethoxy-3-(3-nitrophenyl)acrylate(5.6 g, 21.1 mmol) in methanol (100 ml) and the resulting mixture heatedto reflux overnight. The methanol was then evaporated in vacuo and theresidue diluted with water (150 ml). The solution was washed withdichloromethane (2×80 ml), filtered through a pad of celite and thenacidified by the addition of 3M HCl to pH 3. The mixture wasrefrigerated for 18 h then the precipitated solid was collected byfiltration, washed with water (3×30 ml) and dried in vacuo at 40° C. Theresulting solid was recrystallized from ethyl acetate and heptane togive 2-ethoxy-3-(3-nitrophenyl)acrylic acid as a yellow solid (3.06 g,61%).

¹H NMR (δ, 250 MHz, DMSO-d₆)=1.34 (3H, t, 7 Hz), 4.10 (2H, q, 7 Hz),7.04 (1H, s), 7.69 (1H, t, 7.93 Hz), 8.07-8.22 (2H, m), 8.80 (1H, m),13.25 (1H, brs).

Step 5

A mixture of 2-ethoxy-3-(3-nitrophenyl)acrylic acid (3.06 g, 12.9 mmol),methanol (150 ml) and 5% palladium on activated charcoal (0.60 g, 2 mol%) was hydrogenated at 12-30 psi H₂ atmosphere for 2 hr. The mixture wasthen filtered through celite, the filter cake washed with methanol andthe filtrates concentrated at 40° C. under vacuum to give the product asa foamy solid. This was dissolved in water (100 ml) and the solutionfreeze-dried to give (±)-2-ethoxy-3-(3′-aminophenyl)-propanoic acid (2.7g, 100%) as an off-white solid.

¹H NMR (δ, 250 MHz, DMSO-d₆)=1.07 (3H, t, 7 Hz), 2.6-2.85 (2H, m),3.20-3.38 (1H, m), 3.40-3.60 (1H, m), 3.92 (1H, dd, 5 & 7.7 Hz),6.3-6.45 (3H, m), 7.01 (1H, t, 7.6 Hz).

EXAMPLE 9

Molecular Modelling

Molecular modelling studies were performed using SYBYL software version6.9.1 (Tripos Associates Inc, St Louis, Mo.) running on Silicon Graphicsworkstations. Three-dimensional model of the zwitterion form of 5-ASAwas built from a standard fragments library, and its geometry wassubsequently optimized using the Tripos force field (3) As the pKa ofcompounds are still unknown, the SPARC online calculator was used todetermine the species occurring at physiological pH(7.4)(http://lbmlc2.chem.uga.edu/sparc/index.cfm). Three-dimensionalmodel of ionized compounds were built from a standard fragments library,and their geometry was subsequently optimized using the Tripos forcefield (3) including the electrostatic term calculated from Gasteiger andHückel atomic charges. The method of Powell available in Maximin2procedure was used for energy minimization until the gradient value wassmaller than 0.001 kcal/mol. Å.

The structure of the human PPARγ ligand-binding domain was obtained fromits complexed X-Ray crystal structure with the tesaglitazar (AZ 242)available in the RCSB Protein Data Bank (1I7I) (4,5). Flexible dockingof the compounds into the receptor active site was performed using GOLDsoftware (6). The most stable docking models were selected according tothe best scored conformation predicted by the GoldScore (6) and X-Scorescoring functions (7). The complexes were energy-minimized using thePowell method available in Maximin2 procedure with the Tripos forcefield and a dielectric constant of 4.0 until the gradient value reached0.01 kcal/mol. Å. The anneal function was used defining around theligand a hot region (10 Å) and an interesting region (15 Å).

Results

The molecular modelling receptor docking studies predicted that, ingeneral, the S enantiomer is more active that the R enantiomer, eventhough R enantiomer does show activity also. This phenomenon of oneenantiomer being more biologically active is well known.

As a consequence, the present invention provides a method to resolve thecompounds into enantiomers. The resolution method for compound 32 isshown schematically in FIG. 11.

While not wishing to be bound by theory, it is believed that theS-enantiomers of the compounds will afford higher activity. The resultsof the docking studies are shown in FIGS. 5-10.

EXAMPLE 7 Method of Preparing (±)-2-ethoxy 3-(4′-aminophenyl)-propionicAcid (Compound 39). Enantiomeric Resolution (FIG. 12A and FIG. 12B)

Steps 1 & 2

As per compound 34 steps 1 & 2.

Step 3

2-Hydroxy-3-(4-nitrophenyl)acrylic acid (20 g, 95.6 mmol) was suspendedin DMF (200 ml). Cs₂CO₃ (74.9 g, 229.9 mmol) and diethyl sulphate (26.3ml, 201 mmol) were added and dissolution was observed. After stirringfor 18 hr at 18° C. water (350 ml) and ethyl acetate (250 ml) were addedand the layers separated. The aqueous layer was further extracted withethyl acetate (5×200 ml) then the combined organics were washed withwater (2×200 ml), brine (2×200 ml) and dried over magnesium sulfate. Theorganics were concentrated to dryness to obtain ethyl2-ethoxy-3-(4-nitrophenyl)-acrylate as an orange solid containing 3.6%by mass DMF (27.6 g wet, >100% yield). ¹H NMR (δ, 250 MHz, DMSO-d₆):1.32 (t, 6-H, 2×CH₂CH₃), 4.13 (q, 2-H, CH₂CH₃), 4.30 (q, 2-H, CH₂CH₃),6.99 (s, 1-H, CH═), 8.06 (d, 2-H, CHaromatic), 8.26 (d, 2-H,CHaromatic).

Step 4

Ethyl 2-ethoxy-3-(4-nitrophenyl)acrylate containing 3.6 wt % DMF (26.07g corrected, 98.3 mmol) was dissolved in IMS (500 ml) and a solution ofNaOH (1.44 g, 36.1 mmol) in water (260 ml) was added. The resultingmixture was stirred at ambient temperature for 18 hr then acidified with1M HCl (120 ml) and the resulting solid collected by filtration, washedwith water (2×100 ml) and suction dried on the filter for 30 mins,followed by

Results

Activation of PPARγ results in a cascade of reactions leading to abinding to specific DNA sequence elements termed peroxisome proliferatorresponse elements (PPRE) (7-9).

We investigated PPARγ transcriptional activity by transienttransfections of epithelial cells with the renilla luciferase and PPREplasmids. To evaluate if the new molecules have more efficacy than 5-ASAto stimulate PPARγ activation, we tested these molecules at aconcentration of 1 mM. Effect of the new molecules at a concentration of1 mM was compared to 5-ASA and rosiglitazone, used as positive controlsat optimal concentrations of 30 mM and 10⁻⁵ M respectively. Cells werestimulated with the different molecules during 24 hours.

Analysis of PPARγ activity in transfected HT-29 cells showed that thenew molecules 34, 39, 35 and 40 at 1 mM increased the reporter geneactivity by 4.8±0.71; 2.73±0.31; 2.64±0.46: 3.4±0.97 fold respectively,thereby displaying an activity similar or superior to 5-ASA at 30 mM(2.8±0.7) and rosiglitazone at 10⁻⁵ M (3.17±0.29).

FIG. 2 represents all the results obtained for each molecule assessed in2 or 3 experiments done in triplicate. Reproducibility between thedifferent experiments is good and similar to data described in theliterature.

This study allowed us to identify 4 new molecules having 30 to 50 timesmore efficacy than 5-ASA to activate PPARγ.

EXAMPLE 11

Colon Cancer Cell Growth

The following substances (i.e. 20, 34, 35, 39 and 40) were tested fortheir ability to modulate colon cancer cell growth. For this purpose,three human colon carcinoma cell lines (i.e. HT-29, HT-115 and DLD-1)were used. These cell types were selected on the basis of thecyclooxigenase-2 (COX-2) expression. Indeed, HT-115 cells express abiologically active COX-2, HT-29 cells express a non-functional COX-2isoform, and DLD-1 are COX-2-deficient cells. It is believed that thesemolecules are also active on cells that do not express COX-, and thusthe molecules of the present invention may be used in cells which do notexpress COX-2 for the purposes of treating tumours and otherapplications as herein described.

HT-29 and DLD-1 cells were cultured in McCoy and RPMI1640 mediarespectively, supplemented with 10% fetal bovine serum (FBS), 1%penicillin/streptomycin (P/S) and 50 mg/ml gentamycin. HT-115 werecultured in DMEM medium supplemented with 15% FBS and 1% P/S. Cells weremaintained in a humidified incubator at 37° C., in the presence of 5%CO2.

For cell growth assays, single-cell suspensions were plated at 2×103cells/well (4×103 cells/well for HT115) in 96-well culture dishes inmedium containing 0.5% FBS and allowed to adhere. The non-adherent cellswere then removed, and fresh medium containing 0.5% FBS was added intoeach well. Cells were cultured in the presence or absence of thespecified substances. Each substance was dissolved as a 25 mM stocksolution in culture medium containing 0.5% FBS, and the pH of each stocksolution was adjusted to 7.4, if necessary, with NaOH. Substances wereused at a final concentration ranging from 0.5 to 10 mM.

Cell proliferation was determined by measuring the incorporation of5-bromo-2′-deoxyuridine (BrdU) into DNA using a commercially availablecell proliferation kit (Roche Diagnostics, Monza, Italy). BrdU was addedto the cell cultures during the last 6 hours of incubation, and thelevel of BrdU-positive cells was assessed after 48 h culture byenzyme-linked immunosorbent assay (ELISA), Optical density (OD) wasdetermined at 450 nm using an ELISA reader. Experiments were performedin triplicate and the results are reported as the mean±standarddeviation (SD).

Results

The compounds differed in their ability to inhibit colon cancer cellgrowth. Results are summarized in Table 1 where the percentage ofinhibition of growth of DLD-1 cells by the specified compounds is shown.The substance 20, exhibits a marked anti-proliferative effect in adose-dependent fashion, in each of the three cell lines tested (FIG. 3and FIG. 4). More than 90% of cell growth Inhibition was seen whencompounds were used at a final concentration of 10 mM. The ability ofthe compound 20 to significantly inhibit cell growth was seen when usedat a final concentration of 5 or 10 mM.

The compounds 34 and 39 slightly reduced the cell growth when used athigh doses (10 mM) (FIG. 4), but differences among groups were notstatistically significant. Similarly, no inhibition in cell growth wasseen in cultures added with the substances 35, and 40 (see Table 1).

CONCLUSIONS

This first set of examples of the invention (Example 10) shows theability of four optimized molecules 34, 39, 35 and 40 at concentrationof 1 mM, to increase the PPARγ activity in transfected HT-29 cells,displaying an activity similar or superior to 5-ASA at 30 mM androsiglitazone at 10⁻⁵ M.

The second set examples of the invention (Example 11) shows that thecompounds affect the inhibition of the growth of the colon cancer celllines, HT-29. HT-115 and DLD1 to varying degrees. The compounds differedin their ability to inhibit colon cancer cell growth. The substance 20,exhibits a marked anti-proliferative effect on cell lines tested.

These molecules of the present invention are also active on cells thatdo not express COX-2, and thus the molecules of the present inventionmay be used in cells which do not express COX-2 for the purposes oftreating tumours and other applications as herein described.

OVERALL CONCLUSIONS

The synthesized highest ranking compounds, indicated from modellingstudies, all show an activity similar/superior to that of mesalazine.

REFERENCES

-   1. Dubuquoy, L., E. A. Jansson, S. Deeb, S. Rakotobe, M.    Karoui, J. F. Colombel, J. Auwerx, S. Petterason, and P.    Desreumaux, 2003. Impaired expression of peroxisome    proliferator-activated receptor gamma in ulcerative colitis.    Gastroenterology 124:1265-1276.-   2. Rousseaux C, Lefebvre B, Dubuquoy L, Lefebvre P, Romano O, Auwerx    J, Metzger D, Wahii W, Desvergne B. Naccari G C, Chavatte P, Farce    A, Bulois P, Cortot A, Colombel J F, Desreumaux P. Intestinal    anti-inflammatory effect of 5-amino salicylic acid is dependent on    PPARγ. J Exp Med 2005; 201: 1205-15.-   3. Clark, M. C. R. D. I. V. O., N, 1989. Validation of the General    Purpose Tripos 5.2 Field. J. Comput Chem. 10:982-1012.-   4. Gampe, R. T., Jr., V. G. Montana, M. H. Lambert, A. B.    Miller, R. K. Bledsoe, M. V. Milburn, S. A. Kliewer, T. M. Willson,    and H. E. Xu, 2000. Asymmetry in the PPARγ/RXRalpha crystal    structure reveals the molecular basis of heterodimerization among    nuclear receptors. Mol Cell 5:545-555.-   5. Jones, G., P. Willett, R. C. Glen, A. R. Leach, and R.    Taylor, 1997. Development and validation of a genetic algorithm for    flexible docking. J Mol Biol 267:727-748.-   6. Wang, R., L. Lai, and S. Wang, 2002. Further development and    validation of empirical scoring functions for structure-based    binding affinity prediction. J Comput Aided Mol Des 16:11-26.-   7. Westin, S., R, Kurokawa, R. T. Nolte, G. B. Wisely, E. M.    Mclnerney. D. W. Rose, M. V. Milburn, M. G. Rosenfeld, and C. K.    Glass, 1998. Interactions controlling the assembly of    nuclear-receptor heterodimers and co-activators. Nature 395:199-202.-   8. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G.    Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon,    and et al. 1995. The nuclear receptor superfamily: the second    decade. Cell 83:835-839.-   9. Misra, P., E. D. Owuor, W. Li, S. Yu, C. Qi, K. Meyer, Y. J.    Zhu, M. S. Rao, A. N. Kong, and J. K. Reddy, 2002. Phosphorylation    of transcriptional coactivator peroxisome proliferator-activated    receptor (PPAR)-binding protein (PBP). Stimulation of    transcriptional regulation by mitogen-activated protein kinase. J    Biol Chem 277:48745-48754. Epub 422 Sep 48727.

The invention claimed is:
 1. A method of treating ulcerativerectocolitis in a mammal in need thereof, the method comprisingadministering to the mammal a compound represented by:

or a pharmaceutically acceptable salt or a stereoisomer thereof.
 2. Themethod of claim 1, wherein the mammal is a human.
 3. The method of claim1, wherein the compound is (R)-2-methoxy-3-(4′-aminophenyl)propionicacid.
 4. The method of claim 1, wherein the compound is(S)-2-methoxy-3-(4′-aminophenyl)propionic acid.
 5. The method of claim1, wherein the compound is in an enantiomerically pure R form.
 6. Themethod of claim 1, wherein the compound is in an enantiomerically pure Sform.
 7. A method of treating ulcerative rectocolitis in a mammal inneed thereof, the method comprising administering to the mammal apharmaceutical composition comprising: a compound represented by:

or a pharmaceutically acceptable salt or a stereoisomer thereof; and apharmaceutically acceptable excipient.
 8. The method of claim 7, whereinthe mammal is a human.
 9. The method of claim 7, wherein the compound is(R)-2-methoxy-3-(4′-aminophenyl)propionic acid.
 10. The method of claim7, wherein the compound is (S)-2-methoxy-3-(4′-aminophenyl)propionicacid.
 11. The method of claim 7, wherein the compound is in anenantiomerically pure R form.
 12. The method of claim 7, wherein thecompound is in an enantiomerically pure S form.